1. Field of the Invention
This invention relates to a unique class of compounds which selectively modulate the activity of receptors for the amino acid gamma-aminobutyric acid ("GABA"). Specifically, the modulating compounds act on closed-channel binding sites of GABA.sub.rho receptors, but have little effect on GABA.sub.A and GABA.sub.B receptors.
2. Description of Related Art
Gamma-aminobutyric acid, commonly known as GABA, is an amino acid which serves as the major neurotransmitter in the mammalian central nervous system, particularly the brain. In general, neurotransmitters are responsible for regulating the conductance of ions across neuronal membranes. At rest, the neuronal membrane possesses a potential or membrane voltage of approximately -80 mv, the interior being negative with respect to the exterior of the cell. The potential is the result of ion (K.sup.+, Na.sup.+, Cl.sup.-, organic anions) distribution across the semipermeable neuronal membrane. Neurotransmitters are stored in presynaptic vesicles and are released under the influence of neuronal action potentials. For example, when released into the synaptic cleft, an excitatory chemical transmitter such as acetylcholine will cause membrane depolarization (change of potential from -80 mv to -50 mv or less). This effect is mediated by post-synaptic nicotinic receptors which are activated by acetylcholine, resulting in an increase of membrane permeability to Na.sup.+ ions. The reduced membrane potential triggers neuronal post-synaptic action potential which influence other neurons.
The profound influence of GABA on the central nervous system is related to the presence of GABA receptors in most of the neurons in the brain. GABA regulates the excitability of individual neurons by regulating the conductance of ions across the neuronal membrane. For example, GABA interacts with its recognition site on GABA receptors and causes a related ion channel to open, facilitating the flow of chloride ions down a concentration gradient of the ion channel into the cell. The influx of chloride ions results in the hyperpolarization of the transmembrane potential, thus rendering the neuron less excitable.
GABA receptors have been implicated in the mediation of anxiety, seizures, cognitive function, addictive disorders such as alcholism, and responses to other stresses on the central nervous system. Thus, ligands which enhance or decrease the conductance of ions across the neuronal membrane stimulated by GABA are potentially of significant value in research and therapeutic applications.
Highly common, or "conventional" receptors for GABA are (1) GABA.sub.A, which are ligand gated Cl.sup.- channels, and (2) GABA.sub.B, which are channels active in the regulation of presynaptic K.sup.+ and post-synaptic Ca.sup.2+ levels. Recently, a GABA receptor which is pharmacologically distinct from the GABA.sub.A and GABA.sub.B receptors was expressed in Xenopus oocytes by poly (A).sup.+ RNA isolated from mammalian retinal tissue. Called GABA.sub.ret or GABA.sub.rho (hereafter, GABA.sub.rho), the expressed receptor responded to GABA binding with production of a Cl.sup.- current, indicating that it, like the GABA.sub.A receptors, responds to GABA by mediating Cl.sup.- currents in neuronal membranes. However, although the response of GABA.sub.rho is inhibited by certain Cl.sup.- channel inhibitors which also act on GABA.sub.A. The GABA.sub.rho receptors are insensitive to both the GABA.sub.A antagonist bicuculline ("BIC") and the GABA.sub.B agonist baclofen ("BAC"). As a result, GABA.sub.rho is sometimes described as being a "BIC/BAC insensitive" receptor. In addition, GABA.sub.rho receptors are largely insensitive to steroids which modulate the activity of GABA receptors in both brain and retinal tissue.
For further details concerning the expression and responsiveness of GABA.sub.rho, to various pharmacological agents, those skilled in the art may want to refer to Polenzani, et al., Proc. Natl. Acad. Sci. USA, 88:4318-4322, 1991; Woodward, et al., Mol. Pharmac., 41:89-103, 1992; Woodward, et al., Mol. Pharmac., 42:165-173, 1992; and, Woodward, et al., Mol. Pharmacol., 43: 609-625, 1993, the disclosures of which are incorporated herein for the purpose of providing further background information regarding GABA.sub.rho.
Generally, because GABA.sub.rho is highly expressed in retinal tissue as compared to GABA.sub.A and GABA.sub.B, GABA.sub.rho receptors are likely to play a significant role in visual processing. Ligands which inhibit or enhance the responsiveness of GABA.sub.rho receptors to GABA would, therefore, be reasonably expected to depress or enhance processing of visual stimuli in visual pathways. Further, because GABA.sub.rho receptors appear to respond differently to ligands which affect conventional GABA receptors, ligands which selectively or preferentially bind GABA.sub.rho receptors may have little adverse affect on conventional GABA receptors.
Therefore, not only may ligands which selectively impact GABA.sub.rho receptors have beneficial therapeutic properties, such ligands would also be useful for research purposes, such as probing the function of GABA.sub.rho in vivo, identifying which structural subunits of the receptor render its responsiveness different from that of conventional GABA receptors, and identifying ligands which stimulate specific responses by conventional GABA receptors that are activated or inhibited by presently known ligands for conventional GABA receptors. To date, however, ligands which selectively or preferentially bind GABA.sub.rho receptors have not been identified.
Oocytes of the frog Xenopus laevis are used extensively in the field of molecular neurobiology as an expression system for mammalian neurotransmitter receptors using native poly (A).sup.+ RNA or RNA transcribed in vitro from cloned complementary DNA molecules (i.e., cDNA). A variety of neurotransmitter binding molecules such as G-protein coupled receptors (e.g., GABA.sub.B), ligand-gated receptor/channel complexes (e.g., GABA.sub.A), and voltage-gated channels have been expressed from Xenopus oocytes (for review, see Miledi, et al., Fidia Res. Found.: Neuroscience Award Lectures, Raven Press, 1989, at pp. 57-89). Generally, receptors and channels expressed in Xenopus oocytes retain their binding capacity for, responsiveness to, ligands which they respond to in vivo (see, e.g., Woodward, et al., Mol. Pharmacol., 41:1107-1115, 1992 [GABA.sub.A ]; Woodward, et al., Mol. Pharmacol., 42:165-173, 1992 [GABA.sub.rho ]; Woodward, et al., Mol. Pharmacol., 41:89-103, 1992 [GABA.sub.A and GABA.sub.rho ]; and, Barnard, et al., Neuropharmacology, 26:837-844, 1987 [GABA.sub.A ]).